Solution structure and dynamics of polyubiquitin chains

描述（由申请人提供）：本提案旨在描述真核生物中普遍的蛋白水解信号——多聚素（polybiquitin, polyb）链的溶液结构、动力学和相互作用。泛素-蛋白酶体蛋白水解途径是影响多种重要细胞事件的短寿命蛋白周转的主要调控机制。了解lys48连接的polyb链如何被26S蛋白酶体和其他下游效应分子识别是我们理解调控机制的核心。值得注意的是，通过不同赖氨酸连接的polyb链也作为细胞信号，尽管是调节而不是蛋白水解。尽管有重要证据表明，对不同聚脲链的特异性识别是基于构象决定因素的，但迄今为止，对这些分子在溶液中的构象性质还没有明确的认识。获得这样的信息是绝对必要的，以便发展对不同链如何能够作为特定信号的分子理解。我们将使用核磁共振的方法来确定三维构象的lys48连接的四元- ub链，游离在溶液中，二和四元- ub结合到蛋白酶体受体的分子模拟物，hHR23A和S5a。这将使用来自自旋弛豫和剩余偶极耦合测量的远程定向约束的组合，并辅以顺磁弛豫增强和顺磁标签引入的伪接触位移的距离信息。这些新的核磁共振方法将与基于noe的传统方法以及基于化学位移微扰和溶剂可及性测量的域间或分子间界面的映射相结合。我们将使用一组具有代表性的UBA结构域，以确定各种ub链受体连锁特异性的结构决定因素。结构域运动在polyb链与各种受体相互作用的能力中起着重要作用。我们将引入Ub-Ub界面残基的突变，以确定影响界面稳定性和动力学的决定性因素。我们将在不同的pH值和温度条件下测量二聚脲和四聚脲的蛋白质动力学，以确定结构域间运动的贡献，并表征聚脲链的构象灵活性。

DESCRIPTION (provided by applicant): This proposal is to characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how Lys48-linked polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Remarkably, polyUb chains linked via different lysines also act as cellular signals, although regulatory rather than proteolytic. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of Lys48-linked tetra-Ub chain, free in solution, and of di- and tetra-Ub bound to molecular mimics of the proteasomal receptors, hHR23A and S5a. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. These novel NMR approaches will be used in combination with the conventional methods based on NOEs and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. We will use a representative set of UBA domains in order to identify structural determinants of the linkage specificity of various Ub-chain receptors. Domain motions play essential role in polyUb chain's ability to interact with various receptors. We will introduce mutations in the residues at the Ub-Ub interface in order to identify determining factors contributing to the interface stability and dynamics. We will measure protein dynamics in di-Ub and tetra-Ub under various pH and temperature conditions in order to determine the contribution from interdomain motions and to characterize the conformational flexibility of polyUb chains.